Whole blood collected from volunteer donors for transfusion into recipients is typically separated into components such as red blood cells, white blood cells, platelets, plasma and plasma proteins, using apheresis or other known methods. Each of these separated blood components may be stored individually for later use and are used to treat a multiplicity of specific conditions and disease states. For example, the red blood cell component is used to treat anemia, the concentrated platelet component is used to control bleeding, and the plasma protein component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia.
In cell separation procedures, there are usually some small percentage of other types of cells which are carried over into a separated blood component. When contaminating cells are carried over into a separated component of cells in a high enough percentage to cause some undesired effect, the contaminating cells are considered to be undesirable. For example, white blood cells, are considered undesirable because they may transmit infections such as HIV and CMV may also cause other transfusion-related complications such as transfusion-associated Graft vs. Host Disease (TA-GVHD), alloimmunization and microchimerism.
Whole blood or blood products may also be contaminated with other undesirable pathogens such as viruses and bacteria. The term pathogen as used in this context includes undesirable contaminants such as donor white blood cells, parasites, bacteria and viruses.
Blood screening procedures may miss pathogenic contaminants, and sterilization procedures which do not damage cellular blood components but effectively inactivate all donor white blood cells, infectious viruses and other microorganisms have not heretofore been available.
The use of pathogen reducing agents, which include certain photosensitizers, or compounds which absorb light of defined wavelengths and transfer the absorbed energy to an energy acceptor, have been proposed for reduction of undesirable cells and microorganisms found in blood products or fluids containing blood products. Such photosensitizers may be added to the fluid containing blood or blood components and irradiated.
A number of systems and methods for irradiating pathogens in a fluid with light either with or without the addition of a photosensitizer are known in the art. For example, U.S. Pat. No. 5,762,867 is directed toward a system for activating a photoactive agent present in a body fluid with light emitting diodes (LEDs).
U.S. Pat. No. 5,527,704 is directed toward an apparatus containing LEDs used to activate a fluid containing methylene blue.
U.S. Pat. No. 5,868,695 discloses using LEDs having a red color and emitting light at a wavelength of 690 nm in combination with benzoporphyrin derivative photosensitizers to inactivate red blood cells. As taught in this patent, at a wavelength of 690 nm, red blood cells are essentially transparent to radiation, and as such, the benzoporphyrin derivatives absorb radiation at this wavelength to become activated. Also disclosed in this patent is the use of LEDs having a blue color and emitting light at a peak wavelength of 425 nm to inactivate platelets.
U.S. Pat. No. 5,658,722 discloses irradiating platelets using UVA1 light having an emission peak near 365 nm. This patent teaches that damage to platelets is caused by short UVA<345 nm, and unlike the present invention, calls for removing UVA wavelengths below 345 nm.
Use of UV light which is variably pulsed at a wavelength of 308 nm without the addition of a photosensitizer to inactivate virus in a washed platelet product is taught in an article by Prodouz et al., (Use of Laser-UV for Inactivation of Virus in Blood Products; Kristina Prodouz, Joseph Fratantoni, Elizabeth Boone and Robert Bonner; Blood, Vol 70, No. 2). This article does not teach or suggest the addition of a photosensitizer in combination with light to kill viruses.
One of the features associated with the use of UV light to irradiate red cells has been the propensity of this approach to induce methemoglobin (metHb) formation. In general, metHb levels in normal red cells is less than 1-2%. When levels exceed 10%, symptoms begin to appear in individuals suffering from methemoglobinemia. These include nausea, vomiting, difficulty breathing, discoloration of skin, etc. When levels reach 20-30% severe reactions, including death occurs.
UV light is known to be absorbed by hemoglobin in red cells, primarily through the porphyrin structure of the hemoglobin molecule. Once absorbed, it can result in a change in the oxidation state of the hemoglobin molecule from the Fe2+ (ferrous) to Fe3+ (ferric) state. This occurs via oxidation of the iron chelate. The Fe3+ state is not capable of binding or transporting oxygen. This form of the molecule is known as methemoglobin. Normally, oxidation is reversible through several enzymatic systems in the red cell, which can reduce the Fe3+ state back to Fe2+. Several chemical agents are also known to be able to affect this reduction chemistry. These include methylene blue and riboflavin.
One study on the ability of riboflavin to cause a reduction in hemoglobin from the metHb form back to the normal hemoglobin form was discussed in Dotsch et al. “Comparison of Methylene Blue, Riboflavin, and N-acetylcysteine for the Reduction of Nitric Oxide-induced Methemoglobinemia” (Crit. Care Med 2000, Vol. 28, No. 4, pp. 958-961). The reference showed a concentration effect for this chemistry, with optimal levels being observed at 120 μM.
At the time this study was done, using concentrations of riboflavin greater than 120 μM was not possible due to the solubility limits of riboflavin.
The present invention is directed toward the reduction of pathogens which may be present in red blood cells using Uv light in combination with an endogenous photosensitizer, without extensive formation of methemoglobin or excessive hemolysis of the red blood cells upon illumination with UV light.